Chimeric serine proteases

ABSTRACT

A chimeric serine protease whose protease domain is composeD. of two domain halves (half-sides) with a beta-folded sheet structure, wherein the first domain half corresponds to the first domain half of a first serine protease and the second domain half corresponds to the second domain half of a second serine protease, has improved properties and can be readily crystallized.

This is a divisional of copending application Ser. No. 09/197,801, filedNov. 23, 1998.

BACKGROUND OF THE INVENTION

Human serine proteases and serine proteases from mammals are involved innumerous physiological processes (Barrett, A. J., Methods in Enzymology,Vol. 244 (1994) Academic Press, New York; Twining, S. S., Crit. Revs.Biochem. Mol. Biol. 29 (1994) 315-383). These are essentially proteindigestion, blood coagulation (Davie, E. W. et al., Biochemistry 20(1991) 10363-10370), fertilization (Baba, T., FEBS Letters 27 (1989)296-300), programmed cell death as well as complement activation in theimmune response (Goldberger, G. et al., J. Biol. Chem. 262 (1987)10065-10071). Furthermore serine proteases are known from insect cells(Gay, N. J. et al., Biochim. Biophys. Acta 1132 (1992) 290-296) fromviruses (Allaire, M. et al., Nature 369 (1994) 72-76) as well as fromprokaryotes. Prokaryotic serine proteases are for example subtilisin(Kraut, J., in The Enzymes (Boyer, P. D., ed.) Vol. 3, 547-560 (1971)Academic Press, New York and London), carboxypeptidase II (Liao, D. etal., Biochemistry 31 (1992) 9796-9812) and Streptomyces griseus trypsin(Read, R. J. and James, M. N. G. , J. Mol. Biol. 200 (1988) 523-551).

Blood homoeostasis, the equilibrium between blood coagulation andfibrinolysis is ensured by several very complex systems which mutuallyinfluence each other. In this connection proteases play a role in bloodcoagulation, closure of wounds by fibrin formation as well as infibrinolysis, i.e. clot lysis. After an injury the “injury signal” isamplified by sequential activation (specific proteolysis) of inactiveproenzymes to active enzymes which initiates blood coagulation andensures a rapid closure of wounds. Blood coagulation (haemostasis) canbe initiated by two paths, the intrinsic path in which all proteincomponents are present in the blood, and the extrinsic path in which amembrane protein, the so-called tissue factor, plays a critical role.The molecular mechanism of blood homoeostasis and the components thatare involved in this has been comprehensively described in severalreview articles (Furie, B. et al., Cell 53 (1988) 505-518; Davie, E. W.et al., Biochem. 30 (1991) 10363-10379; Bergmeyer, H. U. (ed.): Methodsof Enzymatic Analysis, Vol. V, chapter 3, 3rd ed., Academic Press, NewYork (1983)).

If the blood homoeostasis becomes unbalanced (blood coagulation versusfibrinolysis), an increased coagulation tendency of the blood can leadto various thrombotic disorders/diseases such as e.g. deep-veinthrombosis, pulmonary embolism, cardiac infarction and stroke (Mustard,J. F. et al., In: Haemostasis and Thrombosis. Bloom, A. L. and Thomas,D. P. (eds), 2nd edition, Churchill-Livingstone, Edinburgh, (1987) pp.618-650). Coagulation disorders with bleeding such as e.g. inhaemophilia A (defective factor VIII) and haemophilia B (defectivefactor IX) can occur as a result of a reduced tendency of the blood tocoagulate.

There is therefore a need for substances which can influence the systemof blood coagulation and fibrinolysis according to the medical needs.Factor VIII or factor IX or recently also factor VII isolated from theblood or produced recombinantly is used for example to treat haemophiliaA and B. tPA (tissUe type plasminogen activator) and streptokinase(bacterial plasminogen activator) are used to lyse clot s for exampleafter cardiac infarction. Antithrombotic substances (Harker, L. A. etal., In: Hemostasis and Thrombosis: Basic Principles and ClinicalPraxis, Colman, R. W. et al., (eds.) 3rd edition, Lippincott, Pa.,(1994) pp. 1638-1660) such as e.g. hirudin (peptide composed of 65 aminoacids, specific thrombin inhibitor; Maraganore, J. M., Thrombosis andHaemostasis 70 (1993) 208-211), heparin (heteroglycan, cofactor ofendogenous inhibitors; Barrowcliffe, T. W. et al., In: Haemostasis andThrombosis. Bloom, A. L. et al. (eds.); 3rd edition,Churchill-Livingstone, Edinburgh (1994) Vol. 2, pp. 1417-1438) and oralvitamin K antagonists (inhibitors of γ-carboxylation; Glu residues ofthe Gla domain; Hirsh, J. et al., In: Hemostasis and Thrombosis, BasicPrinciples and Clinical Praxis, Colman, R. W. et al., (eds.), 3rdedition, Lippincott, Pa., (1994) pp. 1567-1583) are used to inhibitblood coagulation. However, the available substances are often stillvery expensive (protein factors) and/or not ideal with regard to theirmedical application and lead to considerable side effects.

All antithrombotic substances interfere with one or usually even severaltargets within the blood coagulation cascade. The inevitable price paidfor a partial inactivation of the haemostatic system by antithromboticsubstances is an increased risk of bleeding. The orally availablevitamin K antagonists interfere with all vitamin K dependent coagulationfactors such as e.g. the blood plasma proteases FVII, FIX, FX andthrombin which have a Gla domain that is post-translationally modifiedby γ-carboxylation. Consequently this antithrombotic therapy is veryunspecific and influences the intrinsic as well as the extrinsichaemostatic system. Like the vitamin K antagonists, heparin interfereswith several targets within the blood coagulation cascade. Theantithrombotic action is due to an increased inactivation of for examplethrombin, FIXa and FXa by an increased rate of formation of the complexwith the natural inhibitor antithrombin III. Even the specific thrombininhibitor hirudin derived from the leech has failed in clinical studiesdue to frequently occurring bleeding. There is therefore a need for newselective and better tolerated antithrombotic substances with animproved benefit to side effect ratio. In this connection the inhibitionof the FXa mediated activation of prothrombin to thrombin by specificFXa inhibitors appears to be an attractive target.

The search for new modulators (activators, inhibitors) of bloodcoagulation, fibrinolysis and homoeostasis can be carried out byscreening libraries of substances and optionally subsequently improvingan identified lead structure by drug modelling. For this it is necessarythat the serine proteases according to the invention are available in acrystalline form.

Attractive targets within blood homoeostasis are for example theactivated serine proteases thrombin, FVIIa, FIXa, FXa, FXIa, FXIIa,kallikrein (blood coagulation), tPA, urokinase, plasmin (fibrinolysis)and activated protein C (regulatory anticoagulant) and inactiveprecursors (zymogens) thereof. Furthermore the complexes which form byinteraction between a blood plasma protease and cofactor during bloodhomoeostasis such as for example FXa::FVa, FIXa::FVIIIa,thrombin::thrombomodulin, FVII/FVIIa::tissue factor are also of interestas a target.

Serine proteases can be produced recombinantly by biotechnologicalmethods. Examples of this are human tissue plasminogen activator,urokinase and subtilisin. However, it has turned out that the serineproteases isolated from natural sources as well as those producedrecombinantly do not fulfil all requirements with regard to substratespecificity, stability and purity that are needed for therapeuticapplications or when they are used to cleave fusion proteins inbiotechnological production processes. In particular the serineproteases factor Xa and kexin (kex 2) are very unstable. Proteasesisolated from animal and/or human raw materials such as e.g. trypsin,thrombin, factor IXa and factor Xa are problematic for a therapeuticapplication or for an application in a production process fortherapeutics since they may be contaminated with human pathogenic agentssuch as e.g. viruses and/or prions.

Moreover proteases isolated from animal and/or microbial raw materialsare very often additionally contaminated with undesired host cellproteases. For this reason the trypsin from animal raw materials that isused to process insulin is treated withL-1-tosylamide-2-phenyl-ethyl-chloromethyl ketone (TPCK) (Kemmler, W. etal., J. Biol. Chem. 246 (1971) 6786-6791) in order to inhibit thechymotrypsin activity in these preparations. Factor Xa preparations areusually contaminated with thrombin. In the purification of lysylendoproteinase from lysobacter, the α-lytic protease has to be separatedby very complicated process steps.

SUMMARY OF THE INVENTION

A chimeric protein comprising a first sequence and a second sequenceC-terminal to the first sequence and linked to the first sequence by oneor more peptide bonds, the first sequence having the amino acid sequenceof the first catalytic domain half of a first serine protease and thesecond sequence having the amino acid sequence of the second catalyticdomain half of a second serine protease different from the first serineprotease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of factor X, trypsin and the chimericrFXT protease constructed from trypsinogen and FX. FXa parts: shown inblack, trypsin parts: shown in white. Abbreviations: AP, activationpeptide: AA, aromatic amino acid stack domain; CD, catalytic domain;EGF1, epidermal growth factor-like domain; EGF2, epidermal growthfactor-like domain; GLA, domain rich in γ-carboxy-glutamic acidresidues.

FIG. 2: shows the nucleotide and derived amino acid sequence of the FXTbase gene. Additional mutations introduced into the N-terminal FXahalf-side and into the C-terminal trypsin half-side are underlined(FXT-M variant). (The nucleotide sequence is shown in SEQ ID NO:12)

DETAILED DESCRIPTION OF THE INVENTION

Serine proteases contain a catalytic domain having β-folded sheetstructure. The invention concerns a chimeric serine protease whichcontains a protease domain in which the protease domain is composed oftwo domain halves (half-sides) with a β-folded sheet structure (β-barrelarchitecture) and where the first domain half corresponds to the firstdomain half of a first serine protease and the second domain halfcorresponds to the second domain half of a second serine protease.

According to the invention a first domain half is understood as thedomain half which is located N-terminally and the second domain half isunderstood as the domain half that is located C-terminally.

A chimeric serine protease of this invention is a chimera of any twoserine proteases. However, the subgroup of the chymotrypsin family (SeeRawlings, N. D. et al., Methods Enzymol. 244 (1994) 19-61) is preferred.In an embodiment of this invention the first sequence has the amino acidsequence of the first catalytic domain half of a serine proteaseselected from the group consisting of factor VII, factor VIIa, factorIX, factor IXa, factor X, factor Xa, tissue plasminogen activator,urokinase, and plasmin. In a specific embodiment, the first sequence hasthe amino acid sequence of the first catalytic domain half of factor Xa.Preferably the second sequence has the amino acid sequence of the secondcatalytic domain half of trypsin. In a specific embodiment of thisinvention the first sequence has the amino acid sequence of the firstcatalytic domain half of factor Xa and the second sequence has the aminoacid sequence of the second catalytic domain half of trypsin.

In addition to the catalytic domain, a serine protease can haveadditional N-terminal domains. This invention encompasses chimericproteins without these additional domains and chimeric proteins with anadditional domain. In an embodiment of this invention the chimericprotein further comprises an activation peptide sequence N-terminal tothe first sequence. The activation peptide sequence can be a naturallyoccurring or non-naturally occurring activation sequence. It can also bea fragment of a naturally occurring serine protease having activationpeptide activity. Generally such fragments are from 5 to 8 amino acidsin length. In a specific embodiment of this invention the activationpeptide is a human trypsin activation peptide (SEQ ID NO:13). In anotherspecific embodiment it is a truncated trypsin activation peptide, forexample one having the sequence Asp-Asp-Asp-Asp-Lys.

In a specific embodiment of this invention, the chimeric protein has theamino acid sequence shown in FIG. 2.

The chimeric protein of this invention can be made by any conventionalmeans, including chemical synthesis or recombinant techniques. Suchtechniques are well known in the art. When made recombinantly anexpression vector which encodes the chimeric protein and is capable ofexpressing the protein is constructed and then transformed ortransfected into a prokaryotic or eukaryotic host cell. Such techniquesare well known in the art. See for example, Sambrook, J. et al., (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor). Startingwith a known protein sequence one can determine one or more nucleic acidsequences which encode the protein based upon the genetic code (See,e.g. Lewin, Genes, 3d ed. (Wiley 1987) p. 104). This invention providesa process for recombinantly producing the protein of this invention,comprising expressing the protein in a host cell which contains anexpression vector comprising a nucleic acid sequence which codes for theprotein wherein the nucleic acid sequence is under the transcriptionalcontrol of a transcription control element, and isolating the expressedprotein.

It has surprisingly turned out that chimeric serine proteases in whichboth of their protease domain halves are derived from different serineproteases essentially show a mixed substrate and binding activity, P₁specificity being determined by the C-terminal half-side, the P2specificity by the N-terminal half-side and the P3 and P4 specificity bythe N- and/or C-terminal half-side. The combination of two differentcomplete protease domain halves (half-sides) ensures that functionalsubdomains (S1, S2, S3 and S4 binding pocket) can form (Perona, J. J. etal., Protein Science 4 (1995) 337-360).

Furthermore it has turned out that chimeric proteases can be obtainedaccording to the invention which, in contrast to one or both initialproteases, can be more easily crystallized and consequently considerablyfacilitate structural examinations. It is for example known that humanand animal trypsins crystallize well.

The crystallization of trypsin alone or in a complex with an inhibitoris state of the art (see Protein Data Base (PDB); Bernstein, F. C. etal., J. Mol. Biol. 112 (1977) 535-542); Kurinov, I. V. et al., ProteinScience 5 (1996) 752-758; Stubbs, M. et al. FEBS Letters 375 (1995)103-107; Von der Saal, W. et al., “Archiv der Pharmazie” 329 (1996)73-82). Numerous high resolution trypsin structures are known such asfor bovine trypsin (Huber, R. et al., J. Mol. Biol. 89 (1974) 73-101),porcine trypsin (Huang, Q. et al., J. Mol. Biol. 229 (1993) 1022-1036,rat trypsin (Perona, J. J. et al., J. Mol. Biol. 230 (1993) 919-933) andhuman trypsin I (Gaboriaud, C. et al., J. Mol. Biol. 259 (1996)995-1010).

The blood coagulation factor Xa (FXa) is, like thrombin, an extremelyinteresting target for screening, to find substances which modulateblood coagulation and especially those with an antithrombotic effect.

A prerequisite for a specific optimization by structure-based drugdesign of for example a low molecular FXa inhibitor lead structureidentified by primary screening is the preparation of FXa lead structurecomplexes and determination of their spatial structure.

Although the 3D structure of a truncated form of FXa (the Gla FXa) hasrecently been resolved directly (Padmanabhan, K. et al., J. Mol. Biol.232 (1993) 947-966) and indirectly in a complex with the inhibitorDX-9065a (Brandstetter, H. et al., J. Biol. Chem. 271 (1996)29988-29992), the more comprehensive co-crystallization experiments withother FXa inhibitors (lead structures) have previously failed due to theextremely complicated, laborious and poorly reproduciblecrystallization/co-crystallization of FXa. Thus, this invention alsoprovides the chimeric proteins described above in crystalline form.

Surprisingly it was found that a chimeric protease according to theinvention containing or comprising the protease domain 1 of factor Xaand protease domain 2 of trypsin can be renatured and is enzymaticallyactive, and is similar to FXa with regard to substrate specificity(k_(cat)/k_(m)) and, like trypsin, can readily be crystallized in acomplex with a ligand (such as for example with an FXa inhibitor).

Consequently the invention also concerns the use of the chimericproteases according to the invention

i) to screen for modulators (activators and inhibitors) or

ii) to prepare crystals and/or co-crystals composed of chimeric proteaseand modulator.

Such crystals or co-crystals can be used advantageously for X-raystructural analysis and/or structure-based drug design.

Thus, this invention also provides the chimeric proteins describedherein in crystalline form. This invention also provides a process forobtaining crystal structure data comprising crystallizing a chimericprotein according to this invention, performing x-ray crystallography onthe resulting crystalline protein to produce x-ray crystal structuredata, and collecting the x-ray crystal structure data.

This invention also provides a method of identifying a substance havingserine protease inhibiting activity, comprising the following steps: a)contacting the chimeric protein of this invention with a targetsubstance under conditions such that the protein of this invention wouldexhibit serine protease activity in the absence of the target substance;b) detecting whether the serine protease activity of the proteincontacted by the target substance is decreased relative to the serineprotease activity of the protein in the absence of the target substance;and c) if a decrease in serine protease activity is detected,determining that the target substance has serine protease inhibitingactivity. Decreased protease activity in this assay means that thetarget substance has serine protease inhibitory activity.

This invention provides a process for the production of a substance withantithrombotic action, wherein a chimeric serine protease as describedherein is contacted with a target substance; the effect of the targetsubstance on the activity of the said serine protease is determined; inthe case that the target substance inhibits the activity of the saidserine protease, the target substance is isolated and its composition isidentified; and the target substance identified in such a way issynthesized in an amount which is adequate for a therapeuticapplication.

The spatial structure of many serine proteases is described in detail byLesk, A. M. et al., J. Mol. Biol. 258 (1996) 501-537 and Perona, J. J.et al., Protein Science 4 (1995) 337-360. According to them a serineprotease domain is composed of two homologous structures (half sides,protease domain halves) which are presumed to be formed by geneduplication and modification. These two domains (half-sides, proteasedomain halves) are usually packed asymmetrically in the serine proteasesand the catalytic binding site is located between these two domains.Each of these domains has a β-barrel architecture. The domains areusually composed of 6-10 antiparallel β-folded sheet strands which arefolded into a β-barrel (Murzin, A. G. et al., J. Mol. Biol. 236 (1994)1369-1381 and 1382-1400). A. M. Lesk (1996) refers to the N-terminaldomain of these serine proteases as domain 1 and the C-terminal domainas domain 2. Lesk, A. M. et al. (1996) also describe the domaincompositions for some exemplary serine proteases.

Usually domain 1 extends up to about amino acid position 122±5(numbering according to the chymotrypsin numbering of J. Greer, ProteinsStruct. Funct. Genet. 7 (1990) 317-334). Domain 2 begins at about aminoacid position 122±5. The domain border is such that short intermediateregions can indeed exist which can either be allocated to domain 1 or todomain 2.

This invention is also concerned with the use of such chimeric proteasesfor the proteolytic cleavage of proteins.

Factor X

Factor X is a complex glycosylated protease. It belongs mechanisticallyto the serine protease family. FX is synthesized in the liver as aninactive proenzyme (zymogen), secreted into the blood and is activatedwhen required by specific proteolysis. With respect to the proteindomain arrangement the structure of factor X is analogous to that offactor VII, IX and protein C. Furthermore the amino acid sequences ofthese 4 proteases are very homologous (amino acid sequence identity: ca.40%). They are united in a protease subfamily, the factor IX6family.

According to Furie, B. and Furie, B. C. the proteases of the factor IXfamily (factor VIIe, IX, X and protein C) are composed of

a propeptide

a GLA domain

an aromatic amino acid stack domain

two EGF domains (EGF1 and EGF2)

a zymogen activation domain (activation peptide, AP) and

a catalytic protease domain (CD).

Furthermore the blood plasma proteases are post-translationally modifiedduring secretion:

11-12 disulfide bridges

N-glycosylation and/or O-glycosylation (GLA domain and activationpeptide) (Bharadwaj, D. et al., J. Biol. Chem. 270 (1995) 6537-6542,Medved, L. V. et al., J. Biol. Chem. 270 (1995) 13652-13659)

cleavage of the propeptide

γ-carboxylation of Glu residues (GLA domain)

β-hydroxylation of an Asp residue (EGF domains).

After activation of the zymogens (zymogenic form of the protein) byspecific cleavage of one or two peptide bonds (cleavage of an activationpeptide), the enzymatically active proteases are composed of 2 chainswhich, in accordance with their molecular weight are referred to as theheavy and light chain. In the factor IX protease family the two chainsare held together by an inter-molecular disulfide bridge between the EGF2 domain and the protease domain. The zymogen-enzyme transformation(activation) leads to conformation changes within the protease domain.This enables an essential salt bridge required for the protease activityto form between the α-NH₃ ⁺ group of the N-terminal amino acid of theprotease domain and an Asp residue within the FXa protease domain. TheN-terminal region is very critical for this subgroup of serine proteasesand should not be modified. Only then is it possible for the typicalactive site of serine proteases to form with the catalytic triad Ser,Asp and His (Blow, D. M.: Acc. Chem. Res. 9 (1976) 145-152; Polgar, L.:In: Mechanisms of protease action. Boca Raton, Fla. CRC Press, chapter 3(1989)).

The FX activation peptide processing already begins in the cell duringsecretion (first cleavage between the EGF2 domain and the activationpeptide). Then FX is activated to FXa by a second FIXa or FVIIacatalysed cleavage on the membrane in a complex with cofactor FVIIIa ortissue factor (Mann, K. G. et al., Blood 76 (1990) 1-16).

The catalytic domain of FXa is composed of 254 amino acids, is notglycosylated and forms 4 disulfide bridges. It is structurally composedof 2 barrel-like β-folded sheets, the so-called half-sides. The firsthalf-side extends according to the chymotrypsinogen numbering from aminoacid position 195 to 301 and the second half-side extends from aminoacid position 302 to 448 (Greer, J., Proteins Struct. Funct. Genet. 7(1990) 317-334; McLachlan, A. D., J. Mol. Biol. 128 (1979) 49-79; Lesk,A. D. et al., J. Mol. Biol. 258 (1996) 501-537).

The recombinant production of truncated post-translationallynon-modified blood plasma protease variants of the factor IX family(factor VII, IX, X and protein C) comprising the EGF2 domain, activationpeptide (AP) and catalytic domain (CD) by expression of thecorresponding genes in E. coli and subsequent renaturation andactivation of the inactive protease proteins in vitro is describedcomprehensively in PCT/EP97/03027.

Trypsin

The trypsin proteases are formed in the exocrine acinus cells of thepancreas as inactive proenzymes (zymogens), the so-called trypsinogens.Four different trypsinogens (trypsinogen I, II, III and IV) have beenisolated from human pancreatic juice, enzymatically characterized andthe amino acid sequences have been determined. The two trypsinogen genesthat are expressed most strongly TRYI (trypsinogen I) and TRYII(trypsinogen II) are known. They have been isolated by cloning thecorresponding cDNAs (Emi, M. et al., Gene 41 (1986) 305-310). The humantrypsinogen genes TRYI and TRYII code, in accordance with a secretedprotein, for a common signal peptide of 15 amino acids. This is followedby a prosegment that is characteristic for the trypsinogen genes which,in the case of the human trypsinogens I and II, is composed of theN-terminal activation peptide AlaProPheAspAspAspAspLys (SEQ ID NO:13)(Guy, O. et al., Biochem. 17 (1978) 1669-1675). This prosegment isrecognized by enterokinase which is a glycoprotease secreted into thesmall intestine by the mucosa cells of the small intestine and cleavedin the presence of calcium which converts the inactive trypsinogens intotheir active form, the trypsins. Part of the trypsinogen activationoccurs autocatalytically. However, cleavage by enterokinase is more than1000 times faster.

Like factor Xa, the trypsins belong to the family of serine proteases.Activation of the trypsinogens by cleavage of the N-terminal activationpeptide also in this case leads to a conformation change within theprotease domain with involvement of the free N-terminus (formation of anessential salt bridge between the α-NH₃ ⁺ group of the N-terminal aminoacid of trypsin and the Asp194 residue within the protease domain) whichenables formation of the typical active site for serine proteases withthe catalytic triad Ser, Asp and His.

The human trypsinogen I gene (TRYI), which is the most stronglyexpressed, codes for 247 amino acids including a signal sequence of 15amino acids and an activation peptide of 8 amino acids. The maturetrypsin I isoenzyme is thus composed of 224 amino acid residues. Itcontains 10 cysteine residues which form 5 disulfide bridges (Emi, M. etal., Gene 41 (1986) 305-310)). Like factor FXa the catalytic domain oftrypsin is composed structurally of two “barrel-like” β-folded sheets.The first half-side extends according to the chymotrypsinogen numberingfrom amino acid position 16 to 121 and the second half-side from aminoacid position 122 to 246 (Greer, J., Protein Struct. Funct. Genet 7(1990) 317-334; Lesk, A. D. et al., J. Mol. Biol. 258 (1996) 501-537).

The human trypsin isoenzyme I has a sequence homology of 89% to humantrypsin isoenzyme II, a sequence homology of ca. 75% to bovine trypsinand a sequence homology of ca. 43% to the catalytic domain of humanfactor Xa.

Chimeric factor X/trypsin proteases (rFXT)

FX, trypsinogen and the chimeric rFXT protease are shown schematicallyin FIG. 1. The basic version of the hybrid rFXT protease is composed ofthe N-terminal half-side of the catalytic FXa domain from amino acidposition 217 to 320 (amino acid sequence numbering corresponds to thepublication of Kaul, R. K. et al. (Gene 41 (1986) 311-314) and theC-terminal half-side of the catalytic trypsin domain from amino acidposition 127 to 247 (amino acid sequence numbering corresponds to thepublication of Emi, M. et al., Gene 41 (1986) 305-310). The FXT geneadditionally codes for an N-terminal prosegment with the amino acidsequence MHHHHDDDDK (SEQ ID NO:14). It begins with an ATG start codon,this is followed by a poly-His sequence of 4 histidine residues and itends with a truncated trypsin activation peptide (enterokinase cleavagesite). The N-terminal (FXa) and C-terminal half-side (trypsin) of therFXTa base version were constructed to be more trypsin-like or FXa-likeby the introduction of further mutations. The following modificationswere carried out according to the invention (FXa half-side: QE20YN,C27V; trypsin half-side: W141F, YPGK172SSFI, S190A, D217E, V227I andKNTIAANS239DRSMKTR; chymotrypsinogen numbering corresponds to Greer, J.,Proteins Struct. Funct. Genet. 7 (1990) 317-334).

SEQ ID NO: 1-11 primer N1-N11

SEQ ID NO:12 shows the nucleotide sequence of the FXT base gene (accord.to FIG. 2)

SEQ ID NO:13 activation peptide of human trypsin genes I and II

SEQ ID NO:14 prosegment with truncated trypsin activation peptide

The contents of European Patent Application No. 97121232.9, filed Dec.3, 1997 are incorporated herein by reference.

The invention will be better understood by reference to the followingexamples, which are illustrative of the invention but do not limit theinvention as described herein and defined by the claims.

EXAMPLES

Methods

Recombinant DNA Technique

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., (1989) In: Molecular cloning: A laboratory manual. ColdSpring harbor Laboratory Press, Cold Spring Harbor, N.Y. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Protein Determination

The protein concentration of the protease variants was determined bydetermining the optical density (OD) at 280 nm using the molarextinction coefficients calculated on the basis of the amino acidsequence.

Expression Vector

The vector for the expression of the chimeric rFXT proteases is based onthe expression vector pSAM-CORE for core streptavidin. The preparationand description of the plasmid pSAM-CORE is described in WO 93/09144.The core streptavidin gene was replaced by the desired protease variantgene in the pSAM-CORE vector.

Factor Xa

The cloning of the FX gene and the construction of the plasmid pFX-EK-CD(base vector for the construction of the FXT gene) is described indetail in PCT/EP97/03027. The FX expression unit on the plasmidpFX-EK-CD codes for the N-terminal amino acid sequence MHHHHDDDDK (SEQID NO:14—prosegment with a truncated trypsin activation peptide) and thecatalytic domain of human factor Xa.

Example 1 Cloning of the Human Trypsinogen I Gene (Plasmid: pTRYI)

The trypsinogen I cDNA from bp position 61 to 750 coding for trypsinogenI from amino acid position 19 to 247 (cDNA sequence and amino acidsequence numbering according to the publication of Emi, M. et al., (Gene41 (1986) 305-310)) was amplified as template DNA in a polymerase chainreaction (PCR) according to the method of Mullis, K. B. et al., (MethodsEnzymol. 155, (1987) 350-355) using the PCR primers N1 (SEQ ID NO:1) andN2 (SEQ ID NO:2).

              NcoI N1: 5′-AAAAAACCATG GATGATGATGACAAGATCGTTGGG-3′               MetAspAspAspAspLysIleValGly              HindIIIN2: 5′-AAAAAAAAGCTTCATTAGCTATTGGCAGCTATGGTGTTC-3′

and a commercially available human liver cDNA gene bank (vector: LambdaZAP® II) from the Stratagene Company (La Jolla, Calif., U.S.A.). The PCRprimers introduced a singular NcoI cleavage site and an ATG start codonat the 5′ end of the coding region and a singular HindIII cleavage siteat the 3′ end of the coding region.

The ca. 715 bp long PCR product was digested with the restrictionendonucleases NcoI and HindIII and the ca. 700 bp long NcoI/HindIIItrypsinogen I fragment was ligated into the ca. 2.55 kbp longNcoI/HindIII-pSAM-CORE vector fragment after purification by agarose gelelectrophoresis. The preparation and description of the plasmidpSAM-CORE is described in WO 93/09144. The desired plasmid pTRYI wasidentified by restriction mapping and the TRPI cDNA sequence isolated byPCR was checked by DNA sequencing.

Example 2 Construction of the Chimeric Protease Gene FXT (Plasmid: pFXT)

The basic version of the chimeric FX/trypsin protease (name: proteaserFXT; FIG. 2) is composed of a prosegment with the amino acid sequenceMHHHHDDDDK (ATG-start codon, poly-His sequence and a truncated trypsinactivation peptide (enterokinase cleavage site)), the N-terminalhalf-side of the catalytic FXa domain and the C-terminal half-side ofthe catalytic trypsin domain. The N-terminal FXa half-side was made moretrypsin-like by introducing two further modifications (QE20YN, C27V;chymotrypsin numbering according to Greer, J., Proteins Struct. Funct.Genet. 7 (1990) 317-334).

For this purpose the DNA coding for the N-terminal half side of thecatalytic FXa domain from amino acid position 217 to 320 (amino acidsequence numbering according to the publication of Kaul, R. K. et al.(Gene 41 (1986) 311-314) was amplified as template DNA using the PCRprimers N3 (SEQ ID NO:3) and N4 (SEQ ID NO:4)

              NsiIN3: 5′-AAAAAAATGCATCACCACCACGACGATGACGACAAGATCGTGGGAGGC             MetHisHisHisHisAspAspAspAspLysIleValGlyGly       TAcaAcTGCAAGGACGGGGAGgtaCCCTGGCAGGCCCTGCTCATC-3′       TyrAsnCysLysAspGlyGluValProTrpGlnAlaLeuLeuIle               Van91I N4: 5′ AAAAAA CCAGTGGCTGGAGGGGCGGTGGGCAGAGAGGCAGGCGCCACGTTCATGCG-3′

and the plasmid pFX-EK-CD (preparation and description see:PCT/EP97/03027). A DNA sequence coding for the prosegment MHHHHDDDDKwith a singular NsiI cleavage site at the 5′ end was introduced by meansof the 5′ overhanging end of the PCR primer N3. In addition the twodesired mutations QE20YN and C27V were introduced into primer N3. Themutations in the primers are shown by the bases written in lower cases.The FXa DNA was linked to the trypsin DNA sequence by means of the PCRprimer N4. The 5′ overhanging nucleotide sequence of the N4 primer iscomposed of the trypsin DNA sequence from bp position 385 to 413according to the publication of Emi, M. et al. (Gene 41 (1986) 305 to310) with a singular Van91I cleavage site at the 5′ end (shown in boldtype).

The ca. 390 bp long PCR product was digested with the restrictionendonucleases NsiI and Van91I and the ca. 380 bp long NsiI/Van91IN-terminal half-side fragment was purified by agarose gelelectrophoresis.

The DNA coding for the C-terminal half-side of the catalytic trypsindomain from amino acid position 127 to 247 (amino acid sequencenumbering according to the publication of Emi, M. et al., Gene 41 (1986)305-310) was isolated from the plasmid pTRYI (example 1) as a ca. 360 bplong Van91I/HindIII fragment. Afterwards the NsiI/Van91I-FXa half-sidefragment was ligated with a Van91I/HindIII trypsin half side fragmentand inserted into the ca. 2.55 kbp long NsiI/HindIII-pFX-EK-CD vectorfragment (preparation and description see: PCT/EP97/03027) in a threefragment ligation. The desired plasmid pFXT was identified byrestriction mapping and the DNA sequence amplified by PCR was verifiedby DNA sequencing.

Example 3 Construction of the Chimeric Protease Gene FXT-M (Plasmid:pFXT-M)

The C-terminal trypsin half-side of the chimeric rFXT protease was mademore FXa-like by introducing 6 mutations (W141F, YPGK172SSFI, S190A,D217E, V227I and KNTIAANS239DRSMKTR; chymotrypsinogen numberingaccording to Greer, J., Protein Struct. Funct.Genet. 7 (1990) 317-334).

The desired mutations were introduced into the FXT gene by two and threefragment ligations (plasmid pFXT, example 2) using enzymaticallysynthesized DNA fragments (PCR technique) and a chemically synthesizedDNA fragment (adaptor). The DNA adaptor has a singular restrictioncleavage site at the 5′ and 3′ end. It was prepared from 2 complementaryoligonucleotides by annealing (reaction buffer: 12.5 mmol/l Tris-HCl, pH7.0, and 12.5 mmol/l MgCl₂; oligonucleotide concentration: in each case1 pmol/60 ml).

Cloning cleavage Mutation Oligonucleotide sites/fragment length QE20YNN3 see example 2 NsiI/Van91I ca. 380 Bp N4 C27V N3 see example 2NsiI/Van91I ca. 380 Bp N4 W141F N5 Van91I/SapI ca. 283 Bp D217E N6 V227IN7 adaptor SapI/HindIII ca. 71 Bp KNTIAANS239DRSMKT N8 R N5 Van91I/SapIca. 157 Bp YPGK172SSFI N9 N10 SapI/HindIII ca. 198 Bp S190A N11 N5: SEQID NO:5)             Van91I5′-AAAAAACCAGCCACTGGCACGAAGTGCCTCATCTCTGGCTtcGGCAACACTGCGCAGCTCTGGCG-3′N6: (SEQ ID NO: 6)             SapI5′-AAAAAAGCTCTTCCTCCAGGCTTGTTCTTCTGGCACAGCCtTCACCCCAGGAGACAACTCCTTG-3′N7: (SEQ ID NO:7)           SapI5′-AAAAAAGCTCTTCTGGAaTCTACACCAAGGTCTACAACTACGTGAAATGGATTgaccgt                       TyrThrLysValTyrAsnTyrValLysTrpIleAspArg HindIII   tCtatgaaaaCCcgTtaatgAAGCTTTTTTTT-3′    SerMetLysThrArg****** N8: (SEQID NO: 8)          HindIII5′-AAAAAAAAGCTTcattaAcgGGttttcataGaacggtcAATCCATTTCACGTAGTTGTA                      SapI    GACCTTGGTGTAGAtTCCAGAAGAGCTTTTTT-3′ N9:(SEQ ID NO:9)          SapI5′-AAAAAAGCTCTTCCACAGAACATGTTGCTGGTAATgaTgaaggaagAGGAGGCTTCACACTTAGCCTGGC-3′N10: (SEQ ID NO:10)           SapI5′-AAAAAAGCTCTTCCTGTGTGGGCTTCCTTGAGGGAGGCAAGGATgCtTGTCAGGGTGATTCTGGTGG-3′N11: (SEQ ID NO:11)         HindIII5′-AAAAAAAAGCTTCATTAACGGGTTTTCATAGAACGGTCAATCCATTTCACGTAG-3′

The desired intermediate constructs and the pFXT-M final construct wereidentified by restriction mapping. The desired DNA sequence of the FXT-Mmutant gene (final construct) was confirmed by DNA sequencing.

Example 4

a) Expression of the Chimeric FXT Protease Gene in E.coli

In order to express the FXT gene an E. coli K12 strain (e.g. UT5600,Grodberg, J. et al., J. Bacteriol. 170 (1988) 1245-1253) was transformedwith one of the expression plasmids pFXT and PFXT-M (ampicillinresistance) described in examples 2 and 3 and with the lacI^(q)repressor plasmid pUBS520 (Kanamycin resistance, preparation anddescription see: Brinkmann, U. et al., Gene 85 (1989) 109-114).

The UT5600/pUBS520/cells transformed with the expression plasmids pFXTand pFXT-M were cultured at 37° C. up to an optical density at 550 nm(OD₅₅₀) of 0.6-0.9 in a shaking culture in DYT medium (1% (w/v) yeastextract, 1% (w/v) Bacto Tryptone, Difco and 5% NaCl) containing 50-100mg/l ampicillin and 50 mg/l kanamycin and subsequently induced with IPTG(final concentration 1-5 mmol/l). After an induction phase of 4-8 hours(h) at 37° C., the cells were harvested by centrifugation (Sorvall RC-5Bcentrifuge, GS3 rotor, 6000 rpm, 15 min), washed with 50 mmol/l Tris-HClbuffer pH 7.2 and stored at −20° C. until further processing. The cellyield from a 1 l shaking culture was 4-5 g (wet weight).

b) Expression Analysis

The expression of the UT5600/pUBS520/cells transformed with the plasmidspFXT and pFXT-M was analysed. For this purpose cell pellets from in eachcase 1 ml centrifuged culture medium were resuspended in 0.25 ml 10mmol/l Tris-HCl, pH 7.2 and the cells were lysed by ultrasonic treatment(2 pulses of 30 s at 50% intensity) using a Sonifier® Cell Disruptor B15from the Branson Company (Heusenstamm, Germany). The insoluble cellcomponents were sedimented (Eppendorf 5415 centrifuge, 14000 rpm, 5 min)and 1/5 volumes (vol) 5×SDS sample buffer (1×SDS sample buffer: 50mmol/l Tris-HCl, pH 6.8, 1% SDS, 1% mercaptoethanol, 10% glycerol,0.001% bromophenol blue) was added to the supernatant. The insolublecell debris fraction (pellet) was resuspended in 0.3 ml 1×SDS samplebuffer containing 6-8 M urea, the samples were incubated for 5 min at95° C. and centrifuged again. Afterwards the proteins were separated bySDS polyacrylamide gel electrophoresis (PAGE) (Laemmli, U. K., Nature227 (1970) 680-685) and stained with Coomassie Brilliant Blue R dye.

The protease variants synthesized in E. coli were homogeneous and wereexclusively found in the insoluble cell debris fraction (inclusionbodies, IBs). The expression yield was 10-50% relative to the total E.coli protein.

Example 5 Cell Lysis, Solubilization and Renaturation of the ChimericrFXT Proteases

a) Cell Lysis and Preparation of Inclusion Bodies (IBs)

The cell pellet from 3 l shaking culture (ca. 15 g wet weight) wasresuspended in 75 ml 50 mmol/l Tris-HCl, pH 7.2. The suspension wasadmixed with 0.25 g/ml lysozyme and it was incubated for 30 min at 0° C.After addition of 2 mmol/l MgC₂ and 10 mg/ml DNase I (BoehringerMannhein GmbH, catalogue No. 104159) the cells were disruptedmechanically by means of high pressure dispersion in a French® Pressfrom the SLM Amico Company (Urbana, Ill., U.S.A.). Subsequently the DNAwas digested for 30 min at room temperature (RT). 37.5 ml 50 mmol/lTris-HCl pH 7.2, 60 mmol/l EDTA, 1.5 mol/l NaCl, 6% Triton X-100 wasadded to the preparation it was, incubated for a further 30 min at RTand centrifuged in a Sorvall RC-5B centrifuge (GSA Rotor, 12000 rpm, 15min). The supernatant was discarded, 100 ml 50 mmol/l Tris-HCl, pH 7.2,20 mmol/l EDTA was added to the pellet, it was incubated for 30 minwhile stirring at 4° C. and again sedimented. The last wash step wasrepeated. The purified IBs (1.5-2.0 g wet weight, 25-30% dry mass,100-150 mg protease) were stored at −20° C. until further processing.

b) Solubilization and Derivatization of the IBs

The purified IBs were dissolved within 1 to 3 hours at room temperatureat a concentration of 100 mg IB pellet (wet weight)/ml corresponding to5-10 mg/ml protein in 6 mol/l guanidinium-HCl, 100 mmol/l Tris-HCl, 20mmol/l EDTA, 150 mmol/l GSSG and 15 mmol/l GSH, pH 8.0. Afterwards thepH was adjusted to pH 5.0 and the insoluble components were separated bycentrifugation (Sorvall RC-5B centrifuge, SS34 rotor, 16000 rpm, 10min). The supernatant was dialysed for 24 hours at 4° C. against 100vol. 4-6 mol/l guanidinium-HCl pH 5.0.

c) Renaturation

The renaturation of the protease variants solubilized in 6 mol/lguanidinium-HCl and derivatized with GSSG/GSH was carried out at 4° C.by repeated (e.g. 3-fold) addition of 0.5 ml IB solubilisate/derivativein each case to 50 ml 50 mmol/l Tris-HCl, 0.5 mol/l arginine, 20 mmol/lCaCl₂, 1 mmol/l EDTA and 0.5 mmol/l cysteine, pH 8.5 at intervals of 24hours and subsequent incubation for 48 hours at 4° C. After completionof the renaturation reaction the insoluble components were separated byfiltration with a filtration apparatus from the Satorius Company(Göttingen, Germany) equipped with a deep bed filter K 250 from theSeitz Company (Bad Kreuznach, Germany).

d) Concentration and Dialysis of the Renaturation Preparations

The clear supernatant containing protease was concentrated 10-15-fold bycross-flow filtration in a Minisette (membrane type: Omega 10K) from theFiltron Company (Karlstein, Germany) and dialysed for 24 hours at 4° C.against 100 vol. 20 mmol/l Tris-HCl and 50 mmol/l NaCl, pH 7.2 to removeguanidinium-HCl and arginine. Precipitated protein was removed bycentrifugation (Sorvall RC-5B centrifuge, SS34 rotor, 16000 rpm, 20 min)and the clear supernatant was filtered with a Nalgene® disposablefiltration unit (pore diameter: 0.2 mm) from the Nalge Company(Rochester, N.Y., U.S.A.).

Example 6 Purification of the Renatured Inactive Chimeric rFXT Proteases

The inactive rFXT proteases from the renaturation preparations can, ifrequired, be further purified with chromatographic methods which areknown to a person skilled in the art.

Purification of the Chimeric rFXT Proteases by Ion ExchangeChromatography on Q-Sepharose ff

The concentrated renaturation preparation that had been dialysed against20 mmol/l Tris-HCl and 50 mmol/l NaCl, pH 8.0 was applied to aQ-Sepharose ff column (1.5×11 cm, V=20 ml ; loading capacity: 10 mgprotein/ml gel) from the Pharmacia Biotech Company (Freiburg, Germany)(2 column volumes/hour, 2 CV7h) equilibrated with the same buffer and itwas washed with the equilibration buffer until the absorbance of theeluate at 280 nm had reached the blank value of the buffer. The boundmaterial was eluted by a gradient of 50-500 mmol/l NaCl in 20 mmol/lTris-HCl, pH 8.0 (2 CV/h). The proteases were eluted at an NaClconcentration of 100-150 mmol/l. The fractions containing proteases wereidentified by non-reducing and reducing SDS PAGE and the elution peakwas pooled.

Example 7 Activation of the Chimeric rFXT Proteases With Enterokinase

The chimeric rFXT proteases were digested at 25° C. at a concentrationof 0.5 to 2.0 mg/ml and a substrate/protease ratio of 50:1 to 100:1(enterokinase, restriction protease from calf intestine, BoehringerMannheim, Mannheim, Germany, in 50 mmol/l Tris-HCl, pH 8.0. The timecourse of the enzymatic rFXT activation was monitored by activitydetermination with a chromogenic colour substrate (see example 9b) untilcompletion of the digestion (plateau, maximum activation). For thissamples (10 to 100 ml) were taken at intervals of 5-10 minutes from thereaction mixture over a period of up to 2 hours and the generated rFXTaactivity was determined. After reaching the activity plateau theenterokinase digest was purified by affinity chromatography onbenzamidine-Sepharose.

Example 8 Final Purification of the Activated Chimeric rFXTa Proteases

The digestion mixture was applied (2 CV/h) to a benzamidine-SepharoseCL-6B column (1.0×10 cm, V=8 ml; loading capacity: 2-3 mg protein/mlgel) from the Pharmacia Biotech Company (Freiburg, Germany) that hadbeen equilibrated with 20 mmol/l Tris-HCl, 200 mmol/l NaCl, pH 8.0 andwashed with equilibration buffer until the absorbance of the eluate at280 nm reached the blank value for the buffer. The bound material waseluted by 20 mmol/l Tris-HCl, 200 mmol/l NaCl, 10 mmol/l benzamidine, pH8.0 (2 CV/h). The fractions containing rFXTa protease were identified bynon-reducing and reducing SDS PAGE and activity determination.

The serine protease inhibitor benzamidine used for the elution wasremoved by dialysis against 20 mmol/l Tris-HCl, 200 mmol/l NaCl, pH 8.0.

Example 9 Characterization of the Purified rFXT Protease Variants

a) SDS PAGE

Oligomer and aggregate formation by intermolecular disulfide bridgeformation as well as the homogeneity and purity of the renaturedactivated and purified rFXTa proteases were examined by non-reducing(minus mercaptoethanol) and reducing (plus mercaptoethanol) SDS PAGE(Laemmli, UK, Nature 227 (1970) 680-685).

b) Activity Determination, Determination of the Kinetic Constants

The activity of the renatured activated rFXTa proteases was determinedwith the chromogenic substrates Chromozym® X(N-methoxycarbonyl-D-Nle-Gly-Arg-pNA, Boehringer Mannheim GmbH,Mannheim, Cat. No. 789763), Chromozym® U (Bz-β-Ala-Gly-Arg-pNA,Boehringer Mannheim GmbH, Mannheim, Cat. No.836583), Chromozym® PK(Bz-Pro-Phe-Arg-pNA, Boehringer Mannheim GmbH, Mannheim, Cat. No.378445) and Chromozym® TH (Tosyl-Gly-Pro-Arg-pNA, Boehringer MannheimGmbH, Mannheim, Cat. No. 838268) in comparison with recombinant rFXa(rFX-EGF2-AP-CD; preparation and description see PCT/EP97/03027) andnative human trypsin (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany,Cat. No. T6424). Abbreviations: Bz, benzoyl; pNA, p-nitroaniline.

10-100 μl sample was filled up to 200 μl with 190-100 μl 50 mmol/lTris-HCl, 150 mmol/l NaCl, 5 mmol/l CaCl₂, 0.1% PEG 8000, pH 8.0 and 20μl Chromozym® X, U, PK and TH (0.5-40 mmol/l) were added to and measuredagainst a reagent blank value in an ELISA reader at a wavelength of 405nm and RT. The activity and the kinetic constants were determined fromthe linear initial slope according to the Michaelis Menten equation.

kcat (1/s) Km(μM) kcat/Km (1/μM/s) Chromozym ® X(N-methoxycarbonyl-D-Nle-Gly-Arg-pNA) rFXa 215 199 1.1 fFXTa  52  22 2.4trypsin 153  43 3.6 Chromozym ® U (Bz-β-Ala-Gly-Arg-pNA) rFXa  66 1340.5 fFXTa  68  49 1.4 trypsin 225 208 1.1 Chromozym ® PK(Bz-Pro-Phe-Arg-pNA) rFXa  53 265 0.2 fFXTa 119 115 1   trypsin  38  172.2 Chromozym ® TH (tosyl-Gly-Pro-Arg-PNA) rFXa 107 149 0.7 fFXTa  39 23 1.7 trypsin  95  13 7.3 Bz: benzyl BpNA: p-nitroaniline

Example 10 Crystallization of the Chimeric rFXTa Proteases

The activated purified recombinantly produced rFXTa proteases weredialysed for 6 h at 4° C. against 2×100 vol. 5 mmol/l HEPES pH 6.5 andsubsequently concentrated to a concentration of 5 mg/ml in a Centrikon®10 microconcentrator from the Amicon Company (Witten, Germany). Thecrystallization was carried out by vapour diffusion in a sitting drop. 4ml of the FXa specific inhibitor DX-9065a (Katakura, S. et al., Biochem.Biophys. Res. Commun. 197 (1993) 965-972) was added to 4 ml concentratedrFXTa protease (inhibitor concentration: 0.5 mmol/l in 100 mmol/lTris-HCl, 10% polyethylene glycol 6K (PEG 6K), pH 7.0) and equilibratedat 4° C. by means of vapour diffusion in the sitting drop. Crystals grewafter 3-7 days.

Bibliography

Allaire, M. et al., Nature 369 (1994) 72-76

Baba, T., FEBS Letters 27 (1989) 296-300

Barrett, A. J., Methods Enzymol. Vol. 244 (1994) Academic Press, NewYork

Barrowcliffe, T. W. et al., In: Haemostasis and Thrombosis. Bloom, A. L.et al., (eds.), 3rd edition, Churchill-Livingstone, Edinburgh, (1994)Vol. 2,pp.1417-1438

Bergmeyer, H. U. (ed.): Methods of Enzymatic Analysis, Vol. V, chapter3, 3rd ed., Academic Press, New York (1983)

Bernstein, F. C. et al., J. Mol. Biol. 112 (1977) 535-542

Bharadwaj, D. et al., J. Biol. Chem. 270 (1995) 6537-6542

Blow, D. M.: Acc. Chem. Res. 9 (1976) 145-152; Polgar, L.: Mechanism ofprotease action. Boca Raton, Fla., CRC Press, chapter 3 (1989)

Brandstetter, H. et al., J. Biol. Chem. 271 (1996) 29988-29992

Brinkmann, U. et al., Gene 85 (1989) 109-114

Davie, E. W. et al., Biochemistry 30 (1991) 10363-10370

Emi, M. et al. Gene 41 (1986) 305-310

Furie, B. et al., Cell 53 (1988) 505-518

Gaboriaud, C. et al., J. Mol. Biol. 259 (1996) 995-1010

Gay, N. J. et al., Biochim. Biophys. Acta 1132 (1992) 290-296

Goldberger, G. et al., J. Biol. Chem. 262 (1987) 10065-10071

Greer, J., Proteins Struct. Funct. Genet. 7 (1990) 317-334

Grodberg, J. et al., J. Bacteriol. 170 (1988) 1245-1253

Guy, O. et al., Biochem. 17 (1978) 1669-1675

Harker, L. A. et al., In: Hemostasis and Thrombosis: Basic Principlesand Clinical Praxis, Colman, R. W. et al., (eds.), 3rd edition,Lippincott, Pa., (1994) pp. 1638-1660

Hirsh, J. et al., In: Hemostasis and Thrombosis: Basic Principles andClinical Praxis, Colman, R. W. et al., (eds.), 3rd edition, Lippincott,Pa., (1994) pp. 1567-1583

Huang, Q. et al., J. Mol. Biol. 229 (1993) 1022-1036

Huber, R. et al., J. Mol. Biol. 89 (1974) 73-101

Katakura, S. et al., Biochem. Biophys. Res. Commun. 197 (1993) 965-972

Kaul, R. K. et al., Gene 41 (1986) 311-314

Kemmler, W. et al., J. Biol. Chem. 246 (1971) 6786-6791

Kraut, J., In: The Enzymes (Boyer, P. D. ed.) vol. 3, 547-560 (1971)Academic Press, New York and London

Kurinov, I. V. et al., Protein Science 5 (1996) 752-758

Laemmli, U K, Nature 227 (1970) 680-685

Lesk, A. M. et al., J. Mol. Biol. 258 (1996) 501-537

Liao, D. et al., Biochemistry 31 (1992) 9796-9812

Mann, K. G. et al., Blood 76 (1990) 1-16

Maraganore, J. M., Thrombosis and Haemostasis 70 (1993) 208-211

McLachlan, A. D., J. Mol. Biol. 128 (1979) 49-79

Medved, L. V. et al., J. Biol. Chem. 270 (1995) 13652-13659

Mullis, K. B. et al., Methods Enzymol. 155 (1987) 350-355

Murzin, A. G. et al., J. Mol. Biol. 236 (1994) 1369-1381 and 1382-1400

Mustard, J. F. et al., In: Haemostasis and Thrombosis. Bloom, A. L. andThomas, D. P. (eds.), 2nd edition, Churchill-Livingstone, Edinburgh,(1987) pp. 618-650

Padmanabhan, K. et al., J. Mol. Biol. 232 (1993) 947-966

PCT/EP97/03027

Perona, J. J. et al., J. Mol. Biol. 230 (1993) 919-933

Perona, J. J. et al., Protein Science 4 (1995) 337-360

Polgar, L.: In: Mechanisms of protease action. Boca Raton, Fla., CRCPress, chapter 3 (1989)

Protein Data Base (PDB)

Rawlings, N. D. et al., Methods Enzymol. 244 (1994) 19-61

Read, R. J. and James, M. N. G. , J. Mol. Biol. 200 (1988) 523-551

Sambrook, J. et al., (1989) In: Molecular cloning: A laboratory manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Stubbs, M. et al., FEBS Letters 375 (1995) 103-107

Twining, S. S., Crit. Revs. Biochem. Mol. Biol. 29 (1994) 315-383

Von der Saal, W. et al., Archiv der Pharmazie 329 (1996) 73-82

WO 93/09144

15 1 35 DNA Artificial Sequence Description of Artificial SequenceprimerN1 1 aaaaaaccat ggatgatgat gacaagatcg ttggg 35 2 39 DNA ArtificialSequence Description of Artificial Sequenceprimer N2 2 aaaaaaaagcttcattagct attggcagct atggtgttc 39 3 93 DNA Artificial SequenceDescription of Artificial Sequenceprimer N3 3 aaaaaaatgc atcaccaccacgacgatgac gacaagatcg tgggaggcta caactgcaag 60 gacggggagg taccctggcaggccctgctc atc 93 4 56 DNA Artificial Sequence Description of ArtificialSequenceprimer N4 4 aaaaaaccag tggctggagg ggcggtgggc agagaggcaggcgccacgtt catgcg 56 5 64 DNA Artificial Sequence Description ofArtificial Sequenceprimer N5 5 aaaaaaccag ccactggcac gaagtgcctcatctctggct tcggcaacac tgcgagctct 60 ggcg 64 6 65 DNA Artificial SequenceDescription of Artificial Sequenceprimer N6 6 aaaaaagctc ttcctccaggcttgttcttc tgggcacagc cttcacccca ggagacaact 60 ccttg 65 7 91 DNAArtificial Sequence Description of Artificial Sequenceprimer N7 7aaaaaagctc ttctggaatc tacaccaagg tctacaacta cgtgaaatgg attgaccgtt 60ctatgaaaac ccgttaatga agcttttttt t 91 8 91 DNA Artificial SequenceDescription of Artificial Sequenceprimer N8 8 aaaaaaaagc ttcattaacgggttttcata gaacggtcaa tccatttcac gtagttgtag 60 accttggtgt agattccagaagagcttttt t 91 9 70 DNA Artificial Sequence Description of ArtificialSequenceprimer N9 9 aaaaaagctc ttccacagaa catgttgctg gtaatgatgaaggaagagga ggcttcacac 60 ttagcctggc 70 10 67 DNA Artificial SequenceDescription of Artificial Sequenceprimer N10 10 aaaaaagctc ttcctgtgtgggcttccttg agggaggcaa ggatgcttgt cagggtgatt 60 ctggtgg 67 11 54 DNAArtificial Sequence Description of Artificial Sequenceprimer N11 11aaaaaaaagc ttcattaacg ggttttcata gaacggtcaa tccatttcac gtag 54 12 725DNA Homo sapiens 12 atgcatcacc accacgacga tgacgacaag atcgtgggaggccaggaatg caaggacggg 60 gagtgtccct ggcaggccct gctcatcaat gaggaaaacgagggtttctg tggtggaacc 120 attctgagcg agttctacat cctaacggca gcccactgtctctaccaagc caagagattc 180 aaggtgaggg taggggaccg gaacacggag caggaggagggcggtgaggc ggtgcacgag 240 gtggaggtgg tcatcaagca caaccggttc acaaaggagacctatgactt cgacatcgcc 300 gtgctccggc tcaagacccc catcaccttc cgcatgaacgtggcgcctgc ctctctgccc 360 accgcccctc cagccactgg cacgaagtgc ctcatctctggctggggcaa cactgcgagc 420 tctggcgccg actacccaga cgagctgcag tgcctggatgctcctgtgct gagccaggct 480 aagtgtgaag cctcctaccc tggaaagatt accagcaacatgttctgtgt gggcttcctt 540 gagggaggca aggattcatg tcagggtgat tctggtggccctgtggtctg caatggacag 600 ctccaaggag ttgtctcctg gggtgatggc tgtgcccagaagaacaagcc tggagtctac 660 accaaggtct acaactacgt gaaatggatt aagaacaccatagctgccaa tagctaatga 720 agctt 725 13 8 PRT Homo sapiens 13 Ala Pro PheAsp Asp Asp Asp Lys 1 5 14 10 PRT Homo sapiens 14 Met His His His HisAsp Asp Asp Asp Lys 1 5 10 15 5 PRT Homo sapiens 15 Asp Asp Asp Asp Lys1 5

What is claimed is:
 1. A process for recombinantly producing a protein,said protein comprising a first sequence and a second sequenceC-terminal to the first sequence, the first sequence having the aminoacid sequence of the first catalytic domain half of factor Xa and thesecond sequence having the amino acid sequence of the second catalyticdomain half of trypsin, comprising expressing the protein in a host cellwhich contains an expression vector comprising a nucleic acid sequencewhich codes for the protein wherein the nucleic acid sequence is underthe transcriptional control of a transcription control element, andisolating the expressed protein.